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Circulation Research. 2001;88:e38-e47

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(Circulation Research. 2001;88:e38.)
© 2001 American Heart Association, Inc.

UltraRapid Communication

Retinoids Induce Fibroblast Growth Factor-2 Production in Endothelial Cells via Retinoic Acid Receptor {alpha} Activation and Stimulate Angiogenesis In Vitro and In Vivo

Carlo Gaetano1, Alfonso Catalano1, Barbara Illi, Angelina Felici, Saverio Minucci, Roberta Palumbo, Francesco Facchiano, Antonella Mangoni, Salvatore Mancarella, Judith Mühlhauser, Maurizio C. Capogrossi

From the Laboratorio di Patologia Vascolare (C.G., A.C., B.I., A.F., R.P., F.F., A.M., S.M., J.M., M.C.C.), Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; Sezione di Patologia Clinica (A.C.), Dipartimento di Oncologia e Neuroscienze, Facoltà di Medicina e Chirurgia, Università "G. D’Annunzio", Chieti, Italy; and Dipartimento di Oncologia Sperimentale (S.M.), Istituto Oncologico Europeo, Milan, Italy.

Correspondence to Dr Carlo Gaetano, Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail gaetano{at}idi.it


*    Abstract
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 *Abstract
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Abstract—The effect of retinoic acid (RA) on endothelial cells is still controversial and was examined in the present study. In bovine aortic endothelial cells (BAECs), all-trans RA (ATRA) and 9-cis RA (9CRA), but not 13-cis RA (13CRA), induced fibroblast growth factor-2 (FGF-2) production and exhibited a biphasic dose-dependent effect to enhance BAEC proliferation and differentiation into tubular structures on reconstituted basement membrane proteins (Matrigel); both processes were inhibited by FGF-2–neutralizing antibody. The pan RA receptor (RAR)-selective ligand (E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl)-1-propenyl] benzoic acid and the RAR{alpha}-selective ligand 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid stimulated the production of FGF-2, whereas the addition of the RAR{alpha}-antagonist RO 41-5253 inhibited this effect. In BAECs, the forced expression of RAR{alpha}, but not RARß or RAR{gamma}, enhanced FGF-2 production, whereas the RAR{alpha}-dominant negative, {Delta}403, blocked this effect. Furthermore, RAR{alpha} overexpression directly stimulated BAEC differentiation on Matrigel and potentiated the effects of ATRA in this assay. Finally, ATRA-treated BAECs coinjected with Matrigel subcutaneously in mice induced neovascularization within the Matrigel plug, and ATRA also enhanced angiogenesis in the chicken chorioallantoic membrane assay. In conclusion, RA can stimulate endothelial cell proliferation and differentiation in vitro via enhanced RAR{alpha}-dependent FGF-2 production, and it can also induce angiogenesis in vivo. The full text of this article is available at http://www.circresaha.org.


Key Words: endothelial cell • angiogenesis • retinoic acid • retinoic acid receptor • fibroblast growth factor-2


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Retinoic acid (RA) modulates growth and differentiation of a variety of cell types; however, the effect of RA and the role of its receptors on endothelial cell (EC) function are still poorly characterized.1 2 Most studies suggest that RA and its derivatives have antiangiogenic effects; however, proangiogenic effects have also been reported,3 4 5 6 7 8 and RA has been recently described as a potent inducer of microvascular EC differentiation into capillary-like networks in vitro.3 The biological response to RA is mediated by 2 classes of ligand-dependent transcription factors, members of the nonsteroid nuclear hormone receptor superfamily: the RA receptors (RAR{alpha}, RARß, and RAR{gamma}) and the retinoic X receptors (RXR{alpha}, RXRß, and RXR{gamma}). Vitamin A derivatives retain different affinities for specific receptor subtypes, and the activation of receptor complexes by specific ligands increases their affinity for cis-acting RA-responsive elements present in promoter regions of target genes. To gain more insight into the role of RA in ECs, we examined the effects of different retinoids, including all-trans RA (ATRA), a natural retinoid derived from vitamin A, and 9-cis RA (9CRA) and 13-cis RA (13CRA), which are intracellular products of ATRA isomerization on bovine aortic ECs (BAECs). We found that RA can increase EC proangiogenic behavior in vitro and that it can induce angiogenesis in vivo via enhanced RAR{alpha}-dependent fibroblast growth factor-2 (FGF-2) production.


*    Materials and Methods
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 *Materials and Methods
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Retinoids
ATRA, 9CRA, and 13CRA (Sigma Chemical) were dissolved in ethanol (solvent) and stored at 4°C for <1 month before use. (E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl)-1-propenyl] benzoic acid (TTNPB), 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid (Am580), (E)-1-[2,3,5-trimethyl-5,6,7,8-tetrahydro-2-naphtalenyl-1-propenyl] benzoic acid (SR11234), and RAR{alpha} antagonist RO 41-5253 were kindly provided by LaRoche (Hoffman-LaRoche, Nutley, NJ). All experiments were performed under low-light conditions to minimize retinoid photoisomerization.

Cell Culture
BAECs were isolated and characterized as described9 and used between passages 3 through 8. Cells were cultured in DMEM with 10% FCS; however, before each experiment, BAECs were kept in serum-free DMEM for 24 hours, and all subsequent studies were performed with cells in DMEM containing 1% FCS (complete medium).

Proliferation Assay
BAECs were seeded in 96-well plates (2x103 cells/well) in complete medium and treated with 10-10 to 10-6 mol/L retinoids or solvent alone. Viable cell number was determined by trypan exclusion, and cells were counted with a hemacytometer. Blocking experiments were performed by adding either 20 ng/mL of anti-human FGF-2 monoclonal neutralizing antibody ({alpha}FGF-2, R&D System Europe) or 20 ng/mL of control immunoglobulin (IgG1) (R&D System Europe); medium was replaced every 48 hours. Positive control proliferation assays were performed in the presence of 10 ng/mL recombinant human FGF-2 (rhFGF-2, R&D System) and 400 ng/mL {alpha}FGF-2 or an equivalent amount of control immunoglobulins.

Differentiation Assay
Experiments were performed in 24-multiwell plates coated with 200 µL/well of growth factor–reduced reconstituted basement membrane proteins (Matrigel) (Collaborative Research), as previously described.11 BAECs (8x104 cells/well), pretreated for 3 days with 5x10-8 mol/L ATRA or 9CRA, were seeded in Matrigel-precoated plates in complete medium containing retinoids. The number of tubular structures from triplicate wells (10 fields/well) was quantified for each experimental condition at x20 magnification after 18 hours on Matrigel. Blocking experiments were performed by adding either 20 ng/mL {alpha}FGF-2 or 20 ng/mL control immunoglobulins 24 hours before plating. Positive control differentiation assays were performed in the presence of 10 ng/mL rhFGF-2 and 400 ng/mL {alpha}FGF-2 or an equivalent amount of control immunoglobulins.

Growth Factor Immunoassays
FGF-2, transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor, and platelet-derived growth factor-{alpha}ß were measured using an ELISA assay (R&D Systems) according to the manufacturer’s instructions. BAECs plated in 100-mm dishes (106 cells/dish) were cultured in complete medium containing different concentrations of retinoids. Unless indicated otherwise, in all experimental protocols, the supernatant was changed 24 hours before collecting the conditioned medium (CM). CM was stored at -80°C, and before assay, CM aliquots were concentrated 20 times with centricon-3 microconcentrators (Amicon Inc). The protein concentration of each sample was determined by standard Bradford protein assay (Bio-Rad).

Western Blotting
BAECs plated in 100-mm dishes (107 cells/dish) were cultured in complete medium containing 10-7 mol/L retinoids for 5 days. The CM was concentrated 20 times and analyzed as described previously.10 The presence of immune complexes was detected with the enhanced chemoluminescence ECL detection system (Amersham Life Technologies) according to the manufacturer’s instructions. The membrane was exposed to autoradiography films (Hyperfilm HP, Amersham Life Technologies) from 10 seconds to 10 minutes.

Reverse Transcriptase–Polymerase Chain Reaction, Northern Blot, and Actinomycin D Chasing Analyses of FGF-2 Expression
BAECs were plated in 100-mm dishes (106 cells/dish) and cultured in complete medium containing either 5x10-8 mol/L RA or equivalent amounts of solvent. RNA extraction was performed with the RNAeasy kit (Qiagen Inc) according to the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse transcription reaction using Moloney murine leukemia virus reverse transcriptase kit according to the manufacturer’s instructions (Life Technologies). An aliquot (2 µL) of the reverse transcription reaction was subjected to 30 polymerase chain reaction (PCR) cycles (1 minute at 94°C, 1 minute at 56°C, and 1 minute at 72°C) in the presence of 50 pmol of each primer, 1.5 mmol/L MgCl2, 200 mmol/L dATP, dCTP, dGTP, and dTTP, and 2.5 U of AmpliTaq polymerase (Perkin-Elmer). Specific primers (PR1, 5'-TCAAGTTACAACTTCAAGCAG-3'; PR2, 5'-TATACTGCCCAGTTCGTTTC-3') encompassing nucleotide 469 to 689 of the human FGF-2 sequence and ß-actin primers (A, 5'-GTGTTGGCGTAGAGGT-3'; B, 5'-TCATCACCATTGGC- AATGAG-3') as an internal control were used in each reaction. Northern blot analysis was performed as previously described.11 Briefly, total RNA was extracted as described above, and 25 µg was electrophoresed in denaturing agarose gel and blotted to nylon membrane. Hybridization was performed overnight in hybridization buffer containing 106 cpm/mL of 32P-labeled full-length cDNA probe encoding human FGF-2 (kindly provided by M. Presta, Dipartimento di Scienze Biomediche e Biotecnologia, Università di Brescia, Brescia, Italy). Filter was washed at high stringency (0.1x SSC, 0.5% SDS) and exposed from 1 hour to overnight to Kodak Biomax MS films.

Actinomycin D chasing was performed as previously described.12 Assay cells were treated with RA, as described above, for 72 hours before actinomycin D treatment (5 µg/mL). Cells were harvested after 2, 4, and 8 hours of chasing, total RNA was extracted, and specific FGF-2 signal was detected by Northern blot, as described above.

Transient Transfections and ß-Galactosidase Assay
Equal amounts (4 µg) of RAR{alpha}, RARß, RAR{gamma}, RXRß, and RAR{alpha}-dominant negative ({Delta}403) expression vectors and pCMV vector were transfected into BAECs (2x106 cells/100 mm dish) with lipofectamine plus reagent (GIBCO BRL; Life Technologies Ltd) according to manufacturer’s instructions. pCMV ß-gal (1 µg) was added to each transfection for efficiency normalization. ß-Galactosidase activity was measured by direct staining of the transfected cells and by chemoluminescence with Galacto-Light Plus (TROPIX Inc) according to the manufacturer’s instructions. Transfection efficiency was reproducibly about 95% of the total cell population. FGF-2 production was determined by ELISA 72 hours after transfection.

Matrigel Angiogenesis Assay In Vivo
Experimental protocols involving animals have been approved by the Istituto Dermopatico dell’Immacolata Ethical and Biosafety Committee according to the Declaration of Helsinki and the recommendations of the Bioethics Convention of the Council of Europe. The in vivo Matrigel assay was performed as previously described.10 12 Briefly, 1-month-old female Swiss mice (Harlan Nossan) were injected subcutaneously near the abdominal midline with 600 µL Matrigel (Collaborative Research Inc) containing 106 BAECs pretreated either with 5x10-8 mol/L ATRA or with solvent alone for 72 hours before injection. Cells pretreated with ATRA were mixed with Matrigel containing 5x10-8 mol/L ATRA and either 1 µg {alpha}FGF-2 or control immunoglobulins. Additional Matrigel plugs contained ATRA alone, untreated BAECs, or rhFGF-2 (300 ng/mL), the latter mixed either with 3 µg {alpha}FGF-2 or control immunoglobulins. Mice were killed 14 days after injection, and the Matrigel plugs were recovered by surgical dissection. Blood vessels within paraffin-embedded Matrigel plugs12 were quantified in at least 3 central 5-µm sections cut 100 µm from each other. Before evaluation, the histological sections were stained with Masson’s Trichrome (Bio-Optica).

Chicken Chorioallantoic Membrane Assay
The formation of vessels on chick embryo chorioallantoic membrane was assessed as previously described.12 Briefly, fertilized eggs were placed in an incubator at the onset of embryogenesis and kept at 37°C. After 3 days, a square window was opened into the shell, and 3 mL of albumin was removed to detach the developing chorioallantoic membrane from the shell itself. The opening was closed with cellophane tape, and incubation was continued for 4 additional days. At day 8, a hydron inert synthetic polymer (HydroMed Sciences) soaked with 4 µL of ATRA at a concentration ranging from 10-9 to 10-3 mol/L was laid on the chicken chorioallantoic membrane (CAM). After 48 hours (day 10), the CAM was evaluated for the angiogenic response. In each experiment, 5 eggs per group were evaluated.

Statistical Analysis
Data are expressed as mean±SD. Results were analyzed by a one-way ANOVA test, followed by Student’s t test for significance between unpaired mean values. Post hoc tests according to the Student-Newman-Keuls method were used when the ANOVA P value indicated a statistically significant difference among test groups. P<=0.05 was considered statistically significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
RA Effect on BAEC Proliferation and Differentiation
BAECs were cultured in 1% serum and in the presence of ATRA, 9CRA, or 13CRA at concentrations ranging from 10-9 to 10-6 mol/L. After 7 days, both ATRA and 9CRA enhanced BAEC proliferation, with a peak effect at 5x10-8 mol/L (Figure 1ADown). At this time point and concentration, ATRA and 9CRA caused a similar increase in cell number (107±10% and 110±14% over control, respectively; P<0.05) (Figure 1ADown). Interestingly, ATRA and 9CRA at 10-6 mol/L did not stimulate BAEC proliferation, whereas 13CRA showed a very weak stimulatory effect only at 10-8 to 10-6 mol/L (P<0.05 versus control). In a time-course study, the effect of ATRA and 9CRA (5x10-8 mol/L) to increase cell number achieved statistical significance at day 3 (P<0.05 versus control), whereas the effect of 13CRA became statistically significant at day 5 (P<0.05 versus control) (Figure 1BDown).



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  		Figure 1. Effect of RA on BAEC proliferation. A, Dose-dependent effect of ATRA, 9CRA, and 13CRA on BAEC proliferation. Cell number was determined after 7 days of exposure to different concentrations of retinoids (10-9, 10-8, 5x10-8, 10-7, 5x10-7, and 10-6 mol/L) or solvent alone (control). Both ATRA and 9CRA enhanced BAEC proliferation; the peak increase in cell number was achieved at 5x10-8 to 10-7 mol/L. This effect decreased at higher concentrations and was lost at 10-6 mol/L. 13CRA showed a weaker stimulatory effect, which was maximal at 10-6 mol/L. Results are expressed as percent of control, and average cell number in control at day 7 was 40±3x103. This experiment was performed twice in triplicate. B, Time-dependent effect of ATRA, 9CRA, and 13CRA (5x10-8 mol/L) on BAEC proliferation. Both ATRA and 9CRA increased cell number, and this effect achieved statistical significance at day 3. At day 7, average cell number was 84±4x103 and 79±3x103 for ATRA and 9CRA, respectively, whereas cells cultured in the presence of solvent alone reached the average number of 40±2x10.3 In contrast, the effect of 13CRA on BAEC prolifera-tion achieved statistical significance at day 5; it was weaker than that of ATRA and 9CRA, and average cell number at day 7 was 51±2x10.3 Each point represents mean±SD of 3 to 4 experiments performed in triplicate. C, Representative examples of BAEC differentiation. BAEC pretreatment with ATRA and 9CRA (5x10-8 mol/L) for 72 hours induced differentiation into tubular structures after 18 hours on Matrigel. In contrast, the same concentration of 13CRA and solvent alone did not induce tubular structure development. Microphotographs were taken 18 hours after plating on Matrigel at x20 magnification. D, Average effect of RAs on BAEC differentiation. Effects of different concentrations of ATRA, 9CRA, and 13CRA (10-9, 10-8, 5x10-8, 10-7, 5x10-7, and 10-6 mol/L) to induce BAEC differentiation on Matrigel. A dose-dependent enhancement of BAEC differentiation was observed in response to ATRA and 9CRA with a peak effect at 10-7 mol/L. In contrast, tubular structures failed to develop in response to 13CRA or solvent alone. Results represent mean±SD of 3 experiments performed in triplicate.

RA is a well-known inducer of cell differentiation; therefore, it was determined whether it modulated BAEC differentiation into tubular structures (Figure 1CUp). In our experimental conditions, both ATRA and 9CRA exhibited a dose-dependent effect to enhance BAEC differentiation on Matrigel, with a peak effect at 10-7 mol/L. In contrast, 13CRA at any concentration only slightly induced tubular structure formation, and solvent alone did not stimulate tubular structure development (Figure 1DUp).

These results indicate that ATRA and 9CRA were more effective than 13CRA in stimulating BAEC proliferation and differentiation into tubular structures, with the highest activity being in the nanomolar range.

Effect of RA on Cytokine Secretion From BAECs
CM from BAECs treated with ATRA, 9CRA, and 13 CRA (5x10-8 mol/L) was collected at days 1, 3, 4, 5, and 7 and assayed for the presence of different cytokines. At all time points, platelet-derived growth factor-{alpha}ß, TGF-ß1, and vascular endothelial growth factor in CM were undetectable. In contrast, ELISA determination of FGF-2 content (Figure 2ADown) became positive during day 3 of exposure to ATRA and 9CRA and reached the peak of {approx}50 pg/106 cells/24 hours at day 5. CM from BAECs exposed to 5x10-8 mol/L 13CRA or solvent alone revealed considerably lower FGF-2 levels, and the concentration of this cytokine reached the threshold for detection only at some time points (Figure 2ADown). Thereafter, it was determined whether the ability of RA derivatives to enhance FGF-2 secretion was dose-dependent. CM obtained from BAECs treated with ATRA, 9CRA, and 13CRA at concentrations ranging from 10-9 to 10-6 mol/L was collected after 4 days of exposure to each RA isomer and assayed for the presence of FGF-2 (Figure 2BDown). The FGF-2 content in this CM was low at 10-9 mol/L, reached a peak of {approx}40 pg/106 cells/24 hours at 10-7 mol/L, and showed a plateau effect at 10-6 mol/L (Figure 2BDown). In contrast, 13CRA slightly stimulated FGF-2 production only at 10-7 and 10-6 mol/L (Figure 2BDown).



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  		Figure 2. Effect of RA on FGF-2 production from BAECs. A, FGF-2 levels in CM from RA-treated BAECs. FGF-2 production was detectable during day 3 of exposure to ATRA and 9CRA (5x10-8 mol/L) and reached the highest level at day 5. In contrast, CM collected from BAECs exposed to 13CRA (5x10-8 mol/L) did not contain measurable amounts of FGF-2 at days 1 and 3 and became only slightly positive at days 4, 5, and 7. CMs from BAECs exposed to solvent alone contained low levels of FGF-2 at days 3, 4, and 5 and were negative at days 1 and 7. Results represent mean±SD of 3 experiments performed in duplicate. B, Concentration-dependent effect of different RA isomers on FGF-2 secretion. BAECs were treated with different concentrations of ATRA, 9CRA, and 13CRA (10-9, 10-8, 5x10-8, 10-7, 5x10-7, and 10-6 mol/L) for 4 days. FGF-2 levels showed a similar dose-dependent increase in response to ATRA and 9CRA at all concentrations tested. In contrast, a weak effect of 13CRA on FGF-2 secretion became apparent only at 10-7 to 10-6 mol/L. Graph represents the mean±SD of 3 experiments performed in duplicate. C, Western blot analysis of FGF-2 in CM from RA-treated BAECs. Concentrated CM collected at day 5 after exposure to different RAs was assayed by Western blot analysis. It was identified as a predominant FGF-2 18-kDa band in samples treated with ATRA and 9CRA but not in those exposed to 13CRA (5x10-8 mol/L) or solvent. This experiment was repeated three times with similar results. D, Reverse transcriptase–PCR analysis of FGF-2 mRNA. At 48 hours after treatment with 9CRA and ATRA (5x10-8 mol/L), FGF-2 steady-state mRNA level increased 2- to 3-fold. 13CRA (5x10-8 mol/L) only slightly enhanced FGF-2 mRNA level over control. This experiment was repeated 3 times with similar results. E, Northern analysis of FGF-2 mRNA at different time points during treatment with ATRA. BAECs were treated either with 5x10-8 mol/L ATRA for 24, 48, and 72 hours or with solvent for 72 hours. Total RNAs (25 µg) were loaded in each lane. A single FGF-2 mRNA band (6.7 kb) was detected in all samples and exhibited a 3.5- and 6-fold increase compared with solvent alone at 48 and 72 hours, respectively. GAPDH signal was used for normalization. This experiment was repeated twice with similar results. F, Actinomycin D chase analysis of FGF-2 mRNA after treatment with ATRA. BAECs were treated with 5x10-8 mol/L ATRA for 72 hours. RNA synthesis was chased with actinomycin D (5 µg/mL) for an additional 2, 4, and 8 hours. Total RNA was extracted and analyzed for FGF-2 expression by Northern blot. Graph represents the values of the normalized optical density (OD) plotted as percent control at time zero. In the presence of ATRA (dotted line), the FGF-2 mRNA was reduced about 50% versus control (filled line) after 4 hours of chasing. The experiment was repeated 3 times with similar results.

Western blot analysis of concentrated CMs from BAECs treated for 5 days with ATRA and 9CRA but not with 13CRA or solvent alone showed a predominant 18-kDa band, corresponding to the major FGF-2 secreted isoform (Figure 2CUp). The increase in secreted FGF-2 protein was associated with a 2- to 3-fold relative increase in FGF-2 mRNA steady-state level after 48 hours of exposure to ATRA and 9CRA by reverse transcriptase–PCR analysis (Figure 2DUp). Northern analysis was performed on total RNA from BAECs treated with ATRA (5x10-8 mol/L) for 24, 48, and 72 hours. The relative steady-state level of FGF-2 RNA was increased at all time points compared with solvent-treated samples; specifically, the increase was 3-fold at 48 hours and {approx}6-fold at 72 hours (Figure 2EUp). However, in this condition, RNA stability in the presence of actinomycin D was decreased {approx}50% at 4 hours (Figure 2FUp). Taken together, these data indicate that RA enhances FGF-2 mRNA and protein synthesis and that it decreases FGF-2 mRNA stability.

FGF-2 Dependence on RA: Effects on BAECs
To investigate the biological role of FGF-2, {alpha}FGF-2 20 ng/mL was added to cells grown in DMEM with 1% FBS and treated with 5x10-8 mol/L ATRA or 9CRA. In proliferation assays at day 7, the number of cells treated with ATRA and 9CRA and cultured in the presence of the {alpha}FGF-2 was reduced 30±5% and 35±7%, respectively, compared with RA-treated cells cultured with control antibodies (Figure 3ADown). Furthermore, in differentiation assays, the average number of tubular structures for BAECs treated with ATRA or 9CRA (5x10-8 mol/L) in the presence of {alpha}FGF-2 was reduced 70±5% and 75±5%, respectively, compared with cells treated with RAs in the presence of control antibodies (Figure 3BDown). It is noteworthy that in proliferation and differentiation assays, {alpha}FGF-2 failed to completely abolish the effects of ATRA and 9CRA but not those of rhFGF-2. This could be attributable either to an incomplete inhibition of FGF-2 effects by the antibody or to FGF-2–independent mechanisms activated by RA. Nevertheless, the results suggest that at least in part, the effects exerted by RA on EC proliferation and differentiation are attributable to the activation of a FGF-2–dependent autocrine/paracrine loop.



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  		Figure 3. FGF-2 biological activity is required for ATRA and 9CRA enhancement of BAEC proliferation and differentiation. A, Anti-FGF-2 antibody inhibition of RA-dependent enhancement of BAEC proliferation. BAECs were grown for 24 hours in the presence of 10-7 mol/L, ATRA, 9CRA, solvent, or 10 ng/mL of rhFGF-2. Anti–FGF-2 monoclonal antibodies ({alpha}FGF-2) or control immunoglobulins (IgG1) were added (20 ng/mL) to each sample, and cells were cultured for 6 additional days. Molar excess of {approx}5-fold of {alpha}FGF-2 (400 ng/mL) or control immunoglobulins was added to samples treated with rhFGF-2. At day 7, {alpha}FGF-2 significantly (P<0.05) reduced the effect of ATRA, 9CRA, and rhFGF-2 on EC proliferation. Graph represents mean±SD of the number of cells, as percent of control, obtained in 3 independent experiments performed in quadruplicate. In control, cell number at day 7 was 38±5x103. B, Inhibition of BAEC differentiation on Matrigel by anti–FGF-2 antibodies. BAECs were treated for 48 hours with 5x10-8 mol/L ATRA, 9CRA, 13CRA solvent, or rhFGF-2 and then plated on dishes coated with Matrigel either in the presence or absence of {alpha}FGF-2 (20 ng/mL) or control immunoglobulins (IgG1) for 18 hours. Approximately 5-fold molar excess of {alpha}FGF-2 or control immunoglobulins (400 ng/mL) was added to samples treated with rhFGF-2. {alpha}FGF-2 significantly (P<0.001) inhibited the effect of ATRA, 9CRA, and rhFGF-2 on tubular structure development. Results represent mean±SD of 3 experiments performed in duplicate.

FGF-2 Production Is Triggered by Retinoid Receptor–Specific Ligands
RA synthetic analogues with distinct receptor affinities were tested to determine which specific RA receptor subtypes increased FGF-2 production. FGF-2 secretion from BAECs was determined after 3 days of exposure to TTNPB, a pan-RAR ligand,13 or to Am580, an RAR{alpha}-specific ligand,14 at concentrations ranging from 10-10 to 10-6 mol/L. In response to these agonists, FGF-2 secretion increased, the threshold for this effect was observed at 5x10-10 mol/L, and a plateau was achieved at 10-9 mol/L (Figure 4ADown). The pan-RXR ligand SR1123415 alone failed to enhance FGF-2 secretion but still exhibited a strong dose-dependent and synergistic effect on FGF-2 production in conjunction with Am580 (right panel) or TTNPB (left panel) (Figure 4BDown). These experiments suggest that both RAR and RXR activation contributed to RA-dependent stimulation of endogenous FGF-2 production. Furthermore, the biological activity of the RAR{alpha}-antagonist RO 41-5253 was tested, a retinoid that specifically antagonizes the binding of RA to RAR{alpha}, neutralizing its transactivating properties.16 17 18 RO 41-5253 in a dose-dependent manner gradually decreased and finally abolished the effects of ATRA, 9CRA, and Am580 (Figure 4CDown), thus substantiating the requirement of an active RAR{alpha}-dependent pathway in the regulation of FGF-2 production.



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  		Figure 4. Specific retinoid receptor ligands modulate FGF-2 release. A, Effect of different RA derivatives on FGF-2 production. BAECs were treated for 4 days with the pan-RAR ligand TTNPB, the RAR{alpha}-specific ligand Am580, and the pan-RXR ligand SR11234 (10-10, 5x10-10, 10-9, 10-8, 5x10-8, 10-7, and 10-6 mol/L) or with equivalent amounts of solvent. At concentrations of 5x10-10 mol/L and higher, both TTNPB and Am580, but not SR11234, or solvent alone significantly (P<0.001) induced FGF-2 production. Results represent mean±SD of 3 experiments performed in duplicate. B, Cooperative effect of the pan-RAR ligand TTNPB and the RAR{alpha}-specific ligand Am580 with the pan-RXR ligand SR11234 to stimulate FGF-2 release. BAECs were treated for 4 days with either 5x10-8 mol/L TTNPB (left) or 5x10-8 mol/L Am580 (right) and a range of concentrations from 10-9 to 10-6 mol/L of SR11234. Under these conditions, SR11234 had a dose-dependent effect to enhance TTNPB- and Am580-dependent FGF-2 release, reaching levels of FGF-2 production comparable to those of ATRA and 9CRA. Results represent mean±SD of 3 experiments performed in duplicate. C, Inhibitory effect of the anti-hormone RO 41-5253 on RA-dependent FGF-2 release. BAECs were treated for 4 days with ATRA, 9CRA, or Am580 (5x10-8 mol/L) and the RAR{alpha} antagonist RO 41-5253 (10-9, 10-8, 5x10-8, 10-7, 5x10-7, and 10-6 mol/L). RO 41-5253 exhibited a dose-dependent effect to decrease and eventually abolish ATRA-, 9CRA-, and Am580-dependent FGF-2 release. Graph represents mean±SD of 3 experiments performed in duplicate.

Effects of RAR{alpha} Overexpression on FGF-2 Secretion and BAEC Differentiation
The role of specific RA receptor complexes was examined additionally in experiments in which expression vectors for RARs in combination with RXRß were transiently cotransfected in BAECs with a LacZ expression vector, and FGF-2 secretion was determined in the presence of 9CRA, the ligand for both RAR and RXR (Figure 5ADown). Cells were treated 24 hours after transfection for an additional 48 hours with 10-7 mol/L 9CRA. FGF-2 levels, determined by ELISA, were upregulated in the presence of RXRß/RAR{alpha} heterodimer, even in absence of the specific ligand. The addition of 9CRA additionally increased FGF-2 release, whereas RO 41-5253 reduced, but did not completely abolish, FGF-2 secretion in both the presence and absence of 9CRA (Figure 5ADown). RAR{alpha}-, RARß-, and RXRß/RARß-transfected cells produced FGF-2 only in response to 9CRA. In contrast, BAECs transfected with pCMV alone, RAR{gamma}, RXRß/pCMV, and RXRß/RAR{gamma} failed to secrete FGF-2 both in the absence of 9CRA and after 48 hours of exposure to 9CRA. To confirm the role of RAR{alpha} in the induction of FGF-2 production, BAECs were transiently transfected with an expression vector encoding for the RAR{alpha}-dominant negative {Delta}403 (Figure 5BDown).19 In CM collected after 72 hours from transfection, the expression of {Delta}D403 inhibited FGF-2 release by ATRA. Taken together, these results show that an active RAR{alpha} is required for EC production of FGF-2 in response to RA. In additional experiments, the effect of RAR{alpha} overexpression on BAEC differentiation into tubular structures was determined. Cells were treated with ATRA, the RAR{alpha}-specific ligand Am580, the RXR ligand SR11234, or both Am580 and SR11234 and then plated on Matrigel. ATRA, Am580, and SR11234 induced mock-transfected BAEC differentiation, and the effect of Am580 in conjunction with SR1134 was enhanced versus that of either agonist alone and comparable to that of ATRA (Figure 5CDown). RAR{alpha} overexpression enhanced BAEC differentiation in the absence of any agonist and, in response to ATRA, the effect was increased 2-fold versus mock- or pCMV-transfected cells (Figure 5CDown). The expression of the RAR{alpha}-dominant negative {Delta}403 receptor reduced by {approx}30% the number of tubular structures formed in the presence of ATRA versus mock- and pCMV-transfected cells (Figure 5CDown). These results correlated with the capacity of RAR{alpha} and its specific ligand to stimulate FGF-2 production and indicated a direct functional role for this receptor in the regulation of BAEC differentiation.



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  		Figure 5. Effect of overexpression of different retinoid receptors on FGF-2 release from BAECs. A, RAR{alpha}-dependent FGF-2 release. Cells were transfected with expression vectors for different RAR receptors, alone or in combination with an RXRß expression plasmid. BAECs were cultured for 24 hours after transfection and treated with RA for an additional 48 hours before collecting CM for ELISA determination of 48-hour FGF-2 release. 9CRA (10-7 mol/L) enhanced FGF-2 secretion from RAR{alpha}-transfected cells and, to a lower extent, from RARß-transfected BAEC. Production of FGF-2 was abolished by the specific RAR{alpha} antagonist RO 41-5253 (10-6 mol/L). Cells transfected with RAR{gamma} did not secrete FGF-2 in response to 9CRA. Transfection of the heterodimer RXRß/RAR{alpha} enhanced FGF-2 production in the absence of RA, and, on addition of 9CRA, FGF-2 release increased additionally; under both conditions, RO 41-5253 inhibited FGF-2 secretion. Cells transfected with the heterodimer RXRß/RARß secreted FGF-2 only in response to 9CRA to levels comparable to those of RARß alone. Cells transfected either with the heterodimer RXRß/RAR{gamma} or with RXRß alone failed to secrete FGF-2 in basal conditions and in response to 9CRA. Graph shows a representative experiment, and similar results were obtained in 3 independent experiments performed in duplicate. Inset shows that by Western analysis, RAR{alpha}, RARß, RAR{gamma}, and RxRß expression were enhanced in transfected cells versus controls. B, RAR{alpha}-dominant negative {Delta}403 effect on FGF-2 production in response to ATRA. BAECs were transfected with an expression vector for the RAR{alpha}-dominant negative receptor, {Delta}403. After 24 hours, cells were treated with ATRA (10-7 mol/L) for an additional 48 hours before CM collection for quantification of 48-hour FGF-2 release. BAECs transfected with {Delta}403 did not secrete detectable FGF-2 levels in response to ATRA. Graph shows a representative experiment performed in triplicate, and similar results were obtained in 3 additional experiments. C, Effect of RAR{alpha} overexpression on BAEC differentiation on Matrigel. Cells were transfected with RAR{alpha}, {Delta}403, and the control empty expression vector pCMV or were mock-transfected with liposomes alone. At 48 hours after transfection, BAECs were treated for an additional 48 hours with ATRA, Am580, SR11234 (10-7 mol/L), or solvent, as indicated, and then plated on Matrigel-coated dishes. Graph depicts the average number of tubular structures after 18 hours of incubation. All RA derivatives enhanced tubular structure development, albeit at different levels, and the effect of Am580 and SR11234 used together was more marked than that of either agonist alone and comparable to that of ATRA. RAR{alpha} overexpression enhanced tubular structure development, even in the absence of ATRA. Additionally, RAR{alpha}-transfected cells responded to ATRA with a marked increase in tubular structure development versus mock- and pCMV-transfected BAECs. Dominant negative {Delta}403 inhibited the effect of ATRA. Similar results were obtained in 2 experiments performed in triplicate.

RA Induces Angiogenesis In Vivo
The angiogenic properties of RA were evaluated in vivo in 2 different models. In one assay, Matrigel plugs containing anti–FGF-2 antibodies or control immunoglobulins and BAECs pretreated either with ATRA or solvent alone were removed 14 days after subcutaneous injection in mice. Representative pictures of the histological sections show that no vessel formation was detectable when either ATRA alone or BAECs pretreated with solvent were added to the Matrigel (Figure 6ADown, panels 3 and 6). In contrast, a significant angiogenic response was observed in plugs containing ATRA plus ATRA-pretreated BAECs (Figure 6ADown, panel 1) and in positive controls containing rhFGF-2 alone (Figure 6ADown, panel 2). However, the number of vessels was significantly reduced in those Matrigel plugs in which neutralizing FGF-2 antibodies were added to ATRA-pretreated BAECs (panel 4) or rhFGF-2 (panel 5). The average data on the number of blood vessels in Matrigel plugs are shown in Figure 6BDown. In additional experiments, the angiogenic effect of RA in the CAM assay was examined. Previous reports indicated that RA could exert an antiangiogenic effect in the CAM assay.20 However, the evidence that RA could activate angiogenesis in vitro as well as in vivo prompted us to verify its properties also in the CAM assay according to the most recent technical improvements.13 The effects of ATRA were compared with those of solvent or rhFGF-2, which is one of the most powerful inducers of blood vessel growth in this system.13 It was found that 400 pmol/egg of ATRA, corresponding to a concentration of 10-4 mol/L, induced a marked increase of vessel density around the graft, with the highest number of vessels radially converging toward the implant, as occurred in the presence of rhFGF-2 (Figure 6CDown). A semiquantitative evaluation of the number of newly formed blood vessels in 3 independent experiments is shown in the TableDown. No significant angiogenic effects were detected at concentrations of ATRA <10-4 mol/L (data not shown). Taken together, these results show that RA has angiogenic properties in vivo that may be at least in part dependent on the induction of FGF-2 release.



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  		Figure 6. Angiogenic effect of ATRA-activated BAEC angiogenesis in the Matrigel assay in vivo. A, Representative examples of blood vessel formation in Matrigel plugs. Histological analysis of Matrigel plugs shows new blood vessel development in Matrigel containing ATRA (5x10-8 mol/L) and BAECs (10-6 cells) pretreated with ATRA (5x10-8 mol/L) for 72 hours before injection (panel 1). A comparable result was obtained in the positive con-trol containing rhFGF-2 (300 ng/mL) (panel 2). Arrowheads indicate vascular structures containing erythrocytes. A significant angiogenic response in Matrigel containing either ATRA but no cells (panel 3) or BAECs pretreated with solvent (panel 6) was not observed. A significant reduction in the number of vessels was observed when {alpha}FGF-2 was added to Matrigel plugs containing BAECs pretreated with ATRA plus ATRA (panel 4) or rhFGF-2 (panel 5). B, Quantitation of blood vessels in Matrigel plugs. Average results for the experiment shown in Figure 6AUp confirm that Matrigel plugs containing BAECs pretreated with ATRA, as well as positive controls containing rhFGF-2, exhibited an angiogenic response. In contrast, few blood vessels were observed in Matrigel containing either untreated BAECs or ATRA but no cells. Number of vessels was significantly reduced in the presence of neutralizing FGF-2 antibodies. Graph represents mean±SD of 5 mice/group, and 3 different histological sections per plug of {approx}250 µm2 were examined. C, RA stimulates angiogenesis in the CAM assay. Chick embryos, 15 days old, were implanted with sponges embedded with 5 µL of rhFGF-2 (1 µg), ATRA (100 µmol/L), or equivalent amounts of solvent. After 48 hours, the formation of new capillaries around the graft was optically evaluated by stereoscopic microscopy. The pictures show a representative CAM assay, in which the formation of vascular structures surrounding the sponges containing ATRA and rhFGF-2 is evident, with the highest number of vessels radially converging toward the implant. Solvent did not induce an angiogenic response. Similar results were obtained in 3 experiments (see the TableUp).


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  		CAM Assay: Semiquantitative Evaluation


*    Discussion
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 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, it is shown that ATRA, a ligand for RARs, and 9CRA, a ligand for both RARs and RXRs, strongly stimulated BAEC proliferation and differentiation into tubular structures, whereas 13CRA, a retinoid that does not bind receptors, was deficient in these functions. Previous reports have extensively characterized RA as a potent inhibitor of cell proliferation and angiogenesis both in vitro and in vivo21 22 23 ; however, in relatively few instances, it has been shown that retinoids can have a mitogenic effect. This observation has been made in different cell types4 5 27 and also in ECs.6 7 8 The apparent discrepancy on the divergent mitogenic effects of retinoids has been previously examined in primary aortic smooth muscle cells,24 and it has been shown that RAR and RXR activation by ATRA or TTNPB inhibited endothelin-stimulated DNA synthesis and cell replication. In contrast, ATRA alone had an opposite action, activating DNA synthesis, and this effect correlated with increased expression of cyclin D1.25 Interestingly, the peak stimulatory effects of retinoids occurred at concentrations of 10-8 or 10-9 mol/L and decreased at higher concentrations; a comparable biphasic effect in the mitogenic and differentiating ability of retinoids has been shown in the present study. In addition to the mitogenic effect of retinoids, our results show that RAR{alpha} activation induces EC differentiation into tubular structures. This is in agreement with a recent study in which it was shown that RA is a strong inducer of urokinase-type plasminogen activator (u-PA) expression and, via this mechanism, causes ECs to differentiate into tubular structures in fibrin matrices.5 In the present study, we show for the first time to our knowledge that retinoids enhance FGF-2 expression and secretion at mRNA and protein level; because FGF-2 upregulates u-PA,26 it is possible that the increase in this angiogenic growth factor may represent a key effector of the angiogenic response attributable to RAR{alpha} activation.

It is noteworthy that the dose-response curves on the mitogenic and differentiating effects of retinoids shown in the present study demonstrated a biphasic action, with a peak response achieved at 5x10-8 to 10-7 mol/L ATRA and 9CRA, which decreased at 10-6 mol/L. These effects were not paralleled by a decrease in FGF-2 secretion, suggesting that at high concentrations, retinoids inhibit EC proliferation and differentiation despite persistently high FGF-2 levels. Similar effects were previously reported for other growth factors, such as insulin-like growth factor II and TGF-ß1.27 28 Furthermore, we observed that RA did not act by enhancing FGF-2 mRNA stabilization, thus indicating a possible association of FGF-2 increase and the activation of specific RNA synthesis processes, as also described for the u-PA gene.5 In this sense, sequence analysis of human and rat FGF-2 5'-flanking regions revealed the presence of putative RA responsive elements, thyroid hormone response elements, and glucocorticoid response elements, suggesting the possibility that this gene may be responsive to hormone-mediated stimuli (C. Gaetano, unpublished data, January 2000). Furthermore, it has been reported that at least one other member of the FGF family, FGF-3, is sensitive to RA at the transcriptional level.29 However, because the regulation of FGF-2 production occurs at transcriptional and posttranscriptional levels, mechanisms other than transcription may also be involved in the RA-dependent induction of FGF-2 release from ECs. Additional experiments are required to elucidate this point.

Taken together, these in vitro data indicate that under the appropriate conditions, RA exhibits a proangiogenic effect mediated by the functional activation of RAR{alpha}, which, in turn, upregulates FGF-2 production, activating an autocrine/paracrine-positive loop. Previous in vivo studies have shown that the systemic treatment with RA inhibited the formation of new vessels after the implant of tumor cells secreting angiogenic factors into the cornea of rats30 and that, in the CAM assay, RA hindered the development of the embryonic vasculature.31 32 33 Different mechanisms may account for discrepancy between the results of the present and previous studies. The proangiogenic effect of retinoids was observed at nanomolar concentration ranges that activate specific receptor-dependent processes,2 and inhibition of angiogenesis was previously observed at concentrations lower than that. Furthermore, substantial technical differences in the CAM assay may account for the opposite effects reported by us and another laboratory20 22 that examined the effect of retinoids on the development of the embryonic vasculature.

The potential physiological role of the proangiogenic effect of retinoids remains to be determined; however, it is noteworthy that multiple RAR and RXR isotypes and isoforms are highly conserved during evolution and present distinct spatiotemporal patterns of expression in developing embryos and adult tissues.31 Surprisingly, with the exception of RXR{alpha}-null mice,32 33 the genetic inactivation of single RARs did not reveal important cardiovascular morphogenic alterations,34 35 indicating that other receptor types can supply the function. However, data derived from the analysis of receptor double mutants suggested a possible involvement of RAR{alpha} in the organogenesis of the cardiovascular system.36 37 In addition, RAR{alpha} is highly expressed in microvascular ECs3 and in vascular smooth muscle cells.4 These previous studies, together with the results of the present work, suggest a link between RAR{alpha}-mediated induction of FGF-2 and the development of the cardiovascular system.

In conclusion, under the appropriate experimental conditions, RA stimulates EC angiogenic behavior via enhanced, RAR{alpha}-dependent, FGF-2 production and also induces angiogenesis in vivo. The ability of retinoids to modulate angiogenesis may reflect the biological relevance of these hormones in the regulation of EC function during development and may also contribute to the design of novel strategies to induce therapeutic angiogenesis.


*    Acknowledgments
 
This work was partially supported by the European Union (grant BMH4-CT95-1160) and by the Associazione Italiana per la Ricerca sul Cancro. The authors would like to thank Gabriella Ricci and Cinzia Carloni for excellent secretarial assistance.


*    Footnotes
 
Original received July 31, 2000; resubmission received January 18, 2001; accepted February 2, 2001.

1 Both authors contributed equally to this work. Back


*    References
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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